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/lp/springer-journals/prevalence-of-antibodies-to-ro-52-in-a-serologically-defined-ecaiscJVGZ
- Publisher
- Springer Journals
- Copyright
- Copyright © 2009 by Parker et al; licensee BioMed Central Ltd.
- Subject
- Biomedicine; Immunology; Internal Medicine
- eISSN
- 1740-2557
- DOI
- 10.1186/1740-2557-6-2
- pmid
- 19267890
- Publisher site
- See Article on Publisher Site
Abstract
Background: Antibodies against Ro-52 have been described in patients with a broad spectrum of autoimmune disease, most commonly in association with anti-Ro-60 in systemic lupus erythematosus and Sjogrens syndrome. However, in inflammatory myositis anti-Ro-52 is frequently present without anti-Ro-60 and is closely linked to the presence of aminoacyl-tRNA synthetase (aats) antibodies. To date there have been no comprehensive reports on the frequency of anti-Ro- 52 in systemic sclerosis (SSc), a disease characterised by hallmark autoantibodies that occur in non- overlapping subsets. Clinically, each antibody-defined group has a distinct pattern of organ involvement, some featuring myositis. Objectives: To determine the frequency of anti-Ro-52 in serologically defined groups of SSc patients and to investigate a possible link with myositis-associated autoantibodies. Methods: Serum samples from 1010 patients with SSc and 55 and 32 patients with anti-aats and anti-Ku respectively were tested for the presence of anti-Ro-52 using a commercial ELISA. Results: The prevalence of anti-Ro-52 was 15–38% in nine of the eleven sub-groups. There were no significant differences in mean anti-Ro-52 levels in these groups with the exception of that defined by the presence of anti-U1-RNP. In the remaining groups defined by anti-Ro-60 and anti- aats, anti-Ro-52 was present in 92% and 100% respectively. In sera from non-SSc patients with anti- aats, anti-Ro-52 was detected in 64%. Conclusion: Anti-Ro-52 is present throughout the SSc population. It is neither more prevalent in the myositis-associated antibody groups nor does it segregate with any other major SSc-specific autoantibodies. The co-existence of anti-Ro-52 with both anti-Ro-60 and anti-aats is confirmed. duce any specific anti-nuclear antibody staining pattern Introduction Antibodies to the 52 kDa protein Ro-52 were first by indirect immunofluorescence, any precipitin line by described in 1988 in addition to antibodies to Ro-60 and immunodiffusion or electroimmunodiffusion, or positive La in the serum of patients with Sjogrens syndrome (SS) results in enzyme-linked immunosorbent assays (ELISA) [1]. Unlike antibodies to Ro-60 and La, they do not pro- containing native antigen [2]. Anti-Ro-52 is mainly Page 1 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2009, 6:2 http://www.jautoimdis.com/content/6/1/2 detected in the diagnostic laboratory because of the inclu- sented a heterogeneous population including patients sion of recombinant antigen in commercial ELISA and with anti-Th-RNP. All sera were tested for anti-nuclear immunoblotting assays [3]. antibodies by indirect immunofluorescence on HEp-2 cell substrate (Bion inc, Illinois, USA) this was the only Antibodies to Ro-52 have been shown to be present with method used to define the patients with ACA. The pres- anti-Ro-60 (with or without co-existing anti-La) at a high ence of other antibodies was confirmed by counterimmu- frequency in sera from patients with systemic lupus ery- noelectrophoresis and radioimmunoprecipitation as thematosus (SLE) and SS [4,5] and one area of interest has previously described [14,15]. The antibody frequencies centred on their possible pathogenic role in the develop- represented in this study do not reflect the prevalence of ment of congenital heart block, a complication of the neo- each antibody type in the SSc patient population as a natal lupus syndrome [6]. Sera that are monospecific for whole. The samples were selected from a bank of frozen anti-Ro-52 (ie without anti-Ro-60) have also been samples stored according to antibody type with patients described in SS and SLE, but only at a low frequency [2,5]. having no defined specific antibody being over repre- It has been reported that anti-Ro-52 (mainly monospe- sented. cific) is present in a large proportion of patients with autoimmune myositis and is closely associated with the Following initial analysis a number of sera from non-SSc myositis-specific anti-aminoacyl-tRNA synthetase (aats) patients were assessed for presence of anti-Ro-52 to pro- antibodies [7,8]. Neither anti-Ro-60 nor anti-La exhibits vide additional data for analysis of association with anti- this association with anti-aats. This finding has lead to aats and anti-Ku, two under represented antibodies in the anti-Ro-52 being termed a myositis-associated autoanti- selected SSc population. Additional sera tested were from body (MAA) [9,10]. 48 patients with antibodies to aats (36 anti-Jo-1, 8 anti- PL7, 4 anti-PL12) and 32 patients positive for anti-Ku SSc is a heterogeneous autoimmune rheumatic disease of antibodies. unknown aetiology characterised by thickening and fibro- sis of the skin and other organs [11]. Virtually all patients Anti-Ro-52 ELISA have autoantibodies to specific cellular components that Anti-Ro-52 was detected by ELISA using purified recom- binant Ro-52 (Quanta Lite SS-A 52 ELISA, INOVA Diag- are mutually exclusive and correlate with well docu- mented clinical subsets of disease including features of nostics, Inc. San Diego, USA) and was performed in overlap with other connective tissue diseases such as pol- accordance with the manufacturer's instructions using the ymyositis (PM) [12]. In this study we have assessed SSc recommended cut-off of 20 U/ml (also shown to be patients characterised for a range of specific antibodies, appropriate by local validation (data not shown)). and additional groups of patients with antibodies to aats and the connective tissue disease associated antibody Ku Statistical analysis for the presence of anti-Ro-52. There are currently no Statistical analysis was performed using SPSS v 11.0 soft- reports detailing the frequency of anti-Ro-52 in a cohort ware. Significant differences in mean anti-Ro-52 levels for of this size. the eleven SSc antibody groups, and the extra anti-aats and anti-Ku groups, were determined using one-way anal- Methods ysis of variance (ANOVA) with post-hoc Bonferroni cor- Patient samples rection. Significant differences were confirmed with the Serum samples were from 1010 patients diagnosed by an Mann-Whitney U-test. A probability value of < 0.05 was experienced rheumatologist at the Royal Free Hospital, a taken to denote statistical significance in all cases. major tertiary referral centre for SSc, according to the pre- liminary ACR criteria [13]. All SSc patients had been con- Results sented for participation in research studies, approved by Sera from 1010 SSc patients divided into eleven groups the local ethics committee. Antibody groups represented based on antibody serology were tested for the presence of were as follows: anti-centromere (ACA) n = 197, anti- anti-Ro-52. The overall frequency of anti-Ro-52 in this topoisomerase (ATA) n = 210, anti-RNA polymerase III selected population was 27% (Table 1). (ARA) n = 207, anti-fibrillarin (AFA) n = 48, anti-Pm-Scl n = 49, anti-U1-RNP n = 58, anti-Ro-60 n = 13, anti-aats Anti-Ro-52 was present in patients with the major SSc- (4 anti-Jo-1, 1 anti-PL7, 1 anti-PL12) n = 6, and anti-Ku n specific antibodies ACA, ATA and ARA at frequencies of = 5. In addition, two further groups were formed from SSc 28%, 19% and 25% respectively with all three groups hav- patients with no defined antibody (NDA) n = 173, and ing a mean anti-Ro-52 level of 22 U/ml. In smaller groups those whose sera produced an ANA pattern of fine speck- including the minor SSc-specific antibodies AFA, anti-Pm- led nucleoplasmic staining with additional homogeneous Scl and the less specific anti-U1-RNP, anti-Ro-52 was nucleolar staining (fsnu) n = 44. The latter group repre- present at 15%, 33% and 38%. Two further heterogene- Page 2 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2009, 6:2 http://www.jautoimdis.com/content/6/1/2 Table 1: Frequency and mean level of anti-Ro-52 in patients tested Antibody Group Number of patients Anti-Ro-52 positive (%) Mean anti-Ro52 level (U/ml) ACA 197 28 22 AFA 48 15 18 ARA 207 25 22 ATA 210 19 22 fsnu 44 32 26 Anti-aats 6 100 109 Anti-Ku 560 49 NDA 173 28 31 Anti-Pm-Scl 49 33 34 Anti-U1-RNP 58 38 50 Anti-Ro-60 13 92 121 Total SSc 1010 27 28 Anti-aats (non-SSc) 55 64 78 Anti-Ku (non-SSc) 32 28 33 ous groups, those without any of the above antibodies but antibody groups except anti-aats – this small group had producing a speckled nucleoplasmic and nucleolar ANA significantly higher anti-Ro-52 concentrations than all the pattern and known to contain a percentage of anti-Th- remaining groups except anti-Ku. There were no signifi- RNP (fsnu) and, with respect to the RFH cohort as a cant differences in anti-Ro-52 levels for the rest of the whole, a disproportionately large group of patients with groups (figure 1) apart from with the anti-U1-RNP group none of the above antibodies or any unifying ANA pattern which had significantly different levels to the ACA, ATA, (NDA) were found to have frequencies of anti-Ro-52 of ARA, AFA and NDA groups but not the anti-Pm-Scl, anti- 32% and 28% respectively. Finally in the three small cate- Ku and fsnu groups. gories the results were as follows: 13 patients constituted a group where the only identified antibody was anti-Ro- To further investigate the association of anti-Ro-52 with 60, 12/13 (92%) were positive for anti-Ro-52. Anti-Ro-60 the small anti-aats and anti-Ku groups we tested sera from was also present in a further 19 patients included in other an additional 48 patients positive for anti-aats and a fur- serological groups because of the presence of a more spe- ther 32 sera from anti-Ku positive patients, none of whom cific antibody. As with those forming the anti-Ro-60 were classified as having SSc. Sixty four percent of the anti- group, 92% of these were positive for anti-Ro-52. Subse- aats sera and 28% of those with anti-Ku were positive for quent statistical analysis of the remaining ten groups was anti-Ro-52 (Table 1). The mean levels of anti-Ro52 in the not significantly altered by the inclusion or exclusion of anti-aats patients remained significantly higher than those these additional nineteen patients. Sera from six SSc seen overall in the SSc patient groups whereas the anti-Ku patients positive for anti-aats were all positive for anti-Ro- association with anti-Ro-52 was indistinguishable from 52 and three out of five anti-Ku positive sera were positive the SSc groups (with the exception of the anti-Ro-60 and for anti-Ro-52. anti-aats groups). Comparisons of mean anti-Ro-52 values showed the anti- Ro-60 group had significantly higher levels than all other Page 3 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2009, 6:2 http://www.jautoimdis.com/content/6/1/2 ACA AFA ARA ATA f s nu anti- anti- ND A anti- anti- anti- anti- anti- aats Ku Pm Scl U1RN P Ro-60 aats Ku (Non-SSc) An ti bo d y gr oup Anti bo Figure 1 di-Ro es) -52 levels in 11 serologically defined groups of SSc patients and 2 additional non-SSc groups (anti-aats and anti-Ku anti- Anti-Ro-52 levels in 11 serologically defined groups of SSc patients and 2 additional non-SSc groups (anti-aats and anti-Ku antibodies). Boxes show interquartile ranges, lines within the boxes indicate median values and lines outside the boxes indicate maximum and minimum values excluding outliers. (See table 1 for numbers of patients in each group). Page 4 of 6 (page number not for citation purposes) Anti-R -52 (U/ ) o ml Journal of Autoimmune Diseases 2009, 6:2 http://www.jautoimdis.com/content/6/1/2 significant difference in the level and frequency of anti- Discussion The majority of work to elucidate clinical correlations of Ro-52 in these three groups it is clear that there is neither anti-Ro-52 has focused on patients with SLE, SS and an association with any of these antibodies nor, by infer- myositis. Similarly, associations with anti-Ro-60 and anti- ence, the clinical outcomes they predict. aats have been well documented. However, there have been relatively few studies addressing the frequency of As a consequence of the association of anti-Ro-52 with this antibody in SSc and no comprehensive reports assess- anti-aats and other studies that link myositis-specific anti- ing associations of anti-Ro-52 with the specific autoanti- Mi-2 and anti-signal recognition particle (SRP) to anti-Ro- bodies that are characteristic of SSc. Initially it was 52, [9] it was hypothesised that the three antibody groups suggested that there was no reactivity of sera from SSc described as having an increased incidence of muscle patients with recombinant anti-Ro-52 [16]. Subsequently, involvement (AFA, anti-Pm-Scl, anti-U1-RNP) might reports on the presence and frequency of anti-Ro-52 have show an increased association of anti-Ro-52 compared to provided conflicting results [2,5,8,17,18]. In this study we the major SSc-specific antibody groups (ACA, ATA, ARA) analysed sera from a substantial cohort of SSc patients where myositis is not a prominent feature [12]. The results representing the entire spectrum of disease, sub-divided from this study do not support this proposal. into 11 groups by autoantibody profile. Anti-Ro-52 was found to be present at a frequency of at least 15% in all Anti-Pm-Scl is associated with an overlap syndrome in antibody groups tested. The overall frequency of anti-Ro- which up to 80% of SSc patients have inflammatory mus- 52 appears to be greater than previously described [8,18]. cle disease [20]. We did not find any significant difference between the anti-Ro-52 levels in anti-Pm-Scl positive Consistent with previous observations, anti-Ro-52 was patients and the major SSc antibodies. However, Frank et present at high frequency in the group of 13 patients in al reported that the response to Ro-52 was clearly present whom the only identified antibody was anti-Ro-60 [5]. in anti-Pm-Scl patients while it was rare in ACA and ATA The co-existence of anti-Ro-52 with the myositis-specific positive SSc patients. It was suggested that detection of aats antibodies was also confirmed. We were able to anti-Ro-52 may prove useful in discriminating between extend the initial data from a small number of SSc patients in these antibody groups [8]. This view is not patients by testing additional sera positive for anti-aats upheld by our findings. and demonstrating a high degree of association with anti- Ro-52, the results being comparable to previous reports This study also expands the limited data available on anti- [7-9]. As with anti-Ro-60 the reason for the high fre- Ro-52 in AFA positive patients. Myositis has been shown quency of anti-Ro-52 in anti-aats positive sera is unknown to be a prominent feature of patients with this antibody. but it is not due to cross-reactivity between anti-Jo-1 and Keonig et al detected anti-Ro-52 in six of fourteen autoim- Ro-52 [7]. mune myositis patients with AFA and in a previous study from this centre 50% of SSc patients with AFA were shown In the nine other groups of SSc patients the frequency of to have myositis [19,21]. There was no association of anti- anti-Ro-52 varied from 15% in the AFA group to 38% in Ro-52 with AFA, indeed the mean anti-Ro-52 level in the the anti-U1-RNP group. When detected by traditional AFA group was lower than in the non-myositis-associated methods, as described in this study, it is a characteristic antibody groups. feature of SSc that each patient will typically have only one of a series of hallmark autoantibodies that therefore The results obtained for anti-U1-RNP show a bimodal dis- occur in a mutually exclusive fashion. However, a recent tribution for anti-Ro-52 with a number of patients having study employing an addressable laser bead immunoassay extremely high levels of the antibody. This is likely to to analyse autoantibodies in the sera of patients with account for the higher mean anti-Ro-52 value observed autoimmune myositis highlighted unusual antibody for the anti-U1-RNP group and the subsequent significant combinations not previously described [19]. Eventually differences from the majority of the SSc antibodies not the wider application of sensitive multiplex methods seen for any other antibody group except anti-Ro-60 or could confirm whether there is an, as yet, undescribed anti-aats. We were unable to determine any specific rea- overlap between the antibody populations. Currently son to explain this interesting finding. each antibody has been shown to be associated with a dis- ease phenotype and can predict features such as age of Only five anti-Ku positive patients were identified in the onset, extent of skin, and type of organ involvement. The SSc population and three were positive for anti-Ro-52. major SSc-specific autoantibodies ACA, ATA and ARA are Anti-Ku was originally described in Japanese patients with predictive of the clinical manifestations of limited disease SSc/PM overlap, but in the North American population it with minimal internal organ involvement, pulmonary appears to be present in a more SLE-like disease [22] The fibrosis and renal involvement respectively. As there is no anti-Ku positive patients had higher anti-Ro-52 values Page 5 of 6 (page number not for citation purposes) Journal of Autoimmune Diseases 2009, 6:2 http://www.jautoimdis.com/content/6/1/2 systemic lupus erythematosus and Sjogrens syndrome. than the majority of the other SSc antibody groups (figure Arthritis Rheum 1990, 33:349-355. 1) but when an expanded number of non-SSc anti-Ku pos- 5. Peene I, Meheus L, Veys EM, De Keyser F: Diagnostic associations itive sera were analysed there was no significant difference in a large and consecutively identified population positive for anti-SSA and/or anti-SSB: the range of associated diseases in anti-Ro-52 levels to the SSc population (with the excep- differs according to the detailed serotype. Ann Rheum Dis 2002, tion of the anti-aats and anti-Ro-60 groups). 61:1090-1094. 6. Buyon JP, Rupel A, Clancy RM: Neonatal lupus syndromes. Lupus 2004, 13:705-712. Anti-Ro-52 was also present in the two miscellaneous 7. Rutjes SA, Vree Egberts WTM, Jongen P, Hoogen F Van den, Pruijn groups. The NDA and fsnu groups in this study were over GJM, Van Venrooij WJ: Anti-Ro-52 antibodies frequently co- occur with anti-Jo-1 antibodies in sera from patients with idi- represented compared to the Royal Free Hospital cohort opathic inflammatory myopathy. Clin Exp Immunol 1997, overall as it was felt that these were possible candidate 109:32-40. patients for the presence of anti-Ro-52, there being no 8. Frank MB, McCubbin V, Trieu E, Wu Y, Isenberg DA, Targoff IN: The association of anti-Ro52 autoantibodies with myositis and other defining antibody detected. The fsnu group was scleroderma autoantibodies. J Autoimmunity 1999, 12:137-142. known to contain a proportion of anti-Th-RNP positive 9. Brouwer R, Hengstman GJD, Vree Egberts W, Ehrfeld H, Bozic B, Ghirardello A, Grondal G, Hietarinta M, Isenberg D, Kalden JR, Lun- patients but due to inconsistent detection by radioimmu- dberg I, Moutsopoulos H, Roux-Lombard P, Vencovsky A, Wikman A, noprecipitation, this could not be classed as a homogene- Seelig H, van Engelen BGM, van Venrooij WJ: Autoantibody pro- ous population. Anti-Ro-52 was detected in 28% and 32% files in the sera of European patients with myositis. Ann Rheum Dis 2001, 60:116-123. respectively and these values were not statistically differ- 10. Sordet C, Goetz J, Sibilia J: Contribution of autoantibodies to ent from any other group (except anti-Ro-60 and anti- the diagnosis and nosology of inflammatory muscle disease. Joint Bone Spine 2006, 73:646-654. aats). Of note anti-Ro-52 was the only detectable anti- 11. Denton CP, Black CM: Scleroderma – clinical and pathological body in 48 patients in the NDA group. advances. Best Pract Res Clin Rheumatol 2004, 18:271-290. 12. Steen VD: Autoantibodies in systemic sclerosis. Semin Arthritis Rheum 2005, 35:35-42. Conclusion 13. Preliminary criteria for the classification of systemic sclero- The data presented here demonstrate that anti-Ro-52 is sis (scleroderma). Subcommittee for scleroderma criteria of prevalent throughout the SSc population and does not the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Arthritis Rheum 1980, segregate with any of the major SSc-specific autoantibod- 23:581-90. ies. We confirm the association with both anti-Ro60 and 14. Bernstein RM, Bunn CC, Hughes GRV: Identification of antibod- ies to acidic antigens by counterimmunoelectrophoresis. aats antibodies but find no evidence that anti-Ro-52 is Ann Rheum Dis 1982, 41:554-555. found at a higher frequency in SSc patients with myositis- 15. Bunn CC, Denton CP, Shi-Wen X, Knight C, Black CM: Anti-RNA associated antibodies. These findings support the hypoth- polymerases and other autoantibody specificities in systemic sclerosis. Br J Rheumatol 1998, 37:15-20. esis that anti-Ro-52 is a general serum marker with limited 16. Ricchiuti V, Briand JP, Meyer O, Isenberg DA, Pruijn G, Muller S: linkage to a myositis phenotype or other clinical manifes- Epitope mapping with synthetic peptides of 52-kD SSA/Ro tations of SSc. protein reveals heterogeneous antibody profiles in human autoimmune sera. Clin Exp Immunol 1994, 95:397-407. 17. Belfiore N, Rossi S, Bobbio-Pallavicini F, Epis O, Caporali R, Mon- Competing interests tecucco C: Anti-Ro (SS-A) 52 kDa and 60 kDa specificities in undifferentiated connective tissue disease. Joint Bone Spine The authors declare that they have no competing interests. 2000, 67:183-187. 18. Fujimoto M, Shimozuma M, Yazawa N, Kubo M, Ihn H, Sato S, Tamaki Authors' contributions T, Kikuchi K, Tamaki K: Prevalence and clinical relevance of 52- kDa and 60-kDa Ro/SS-a autoantibodies in Japanese patients CB and RB designed and coordinated the study. JP per- with systemic sclerosis. Ann Rheum Dis 1997, 56:667-670. formed the assays, the statistical analysis and drafted the 19. Koenig M, Fritzler MJ, Targoff IN, Troyanov Y, J-C Senecal: Hetero- manuscript. All authors contributed to the final revision geneity of autoantibodies in 100 patients with autoimmune myositis: insights into clinical features and outcomes. Arthritis of the manuscript. Research Therapy 2007, 9:R78. 20. Marguerie C, Bunn CC, Copier J, Bernstein RM, Gilroy JM, Black CM, So AK, Walport MJ: The clinical and immunogenetic features Acknowledgements of patients with autoantibodies to the nucleolar antigen PM- We thank Richard Morris for advice with statistics. Scl. Medicine 1992, 71:327-336. 21. Tormey VJ, Bunn CC, Denton CP, Black CM: Anti-fibrillarin anti- References bodies in systemic sclerosis. Rheumatology 2001, 40:1157-1162. 22. Mimori T: Clinical significance of anti-Ku autoantibodies – a 1. Ben-Chetrit E, Chan EKL, Sullivan KF, Tan EM: A 52-kD protein is serological marker of overlap syndrome? Intern Med 2002, a novel component of the SS-A/Ro antigenic particle. J Exp 41:1096-1098. Med 1988, 167:1560-1571. 2. Peene I, Meheus L, De Keyser S, Humbel R, Veys EM, De Keyser F: Anti-Ro-52 reactivity is an independent and additional serum marker in connective tissue disease. Ann Rheum Dis 2002, 61:929-933. 3. McCauliffe DP, Wang L, Satoh M, Reeves WH, Small D: Recom- binant 52 kDa Ro (SSA) ELISA detects autoantibodies in Sjogrens syndrome that go undetected by conventional serologic assays. J Rheumatol 1997, 24:860-866. 4. Ben-Chetrit E, Fox RI, Tan EM: Dissociation of immune responses to the SS-A (Ro) 52-kd and 60-kd polypeptides in Page 6 of 6 (page number not for citation purposes)
Journal
Journal of Autoimmune Diseases – Springer Journals
Published: Mar 6, 2009